Abstract
Redox conditions change in events such as immune and platelet activation, and during viral infection, but the biochemical consequences are not well characterized. There is evidence that some disulfide bonds in membrane proteins are labile while others that are probably structurally important are not exposed at the protein surface. We have developed a proteomic/mass spectrometry method to screen for and identify non-structural, redox-labile disulfide bonds in leucocyte cell-surface proteins. These labile disulfide bonds are common, with several classes of proteins being identified and around 30 membrane proteins regularly identified under different reducing conditions including using enzymes such as thioredoxin. The proteins identified include integrins, receptors, transporters and cell–cell recognition proteins. In many cases, at least one cysteine residue was identified by mass spectrometry as being modified by the reduction process. In some cases, functional changes are predicted (e.g. in integrins and cytokine receptors) but the scale of molecular changes in membrane proteins observed suggests that widespread effects are likely on many different types of proteins including enzymes, adhesion proteins and transporters. The results imply that membrane protein activity is being modulated by a ‘redox regulator’ mechanism.
Highlights
Membrane proteins that reside on the cell surface of leucocytes contain many cysteine (Cys) residues that mainly exist in an oxidized redox state as disulfide bonds
Structural disulfide bonds are usually buried inside the core of a protein or protein domain such as those found in the core of the immunoglobulin (Ig) fold
In order to screen the entire cell surface for proteins that contain redox-labile disulfide bonds, we developed a proteomics workflow based upon subjecting the cells to mild reducing conditions comparable with those expected during an immune response [11] and differentially labelling Cys residues with thiol-modifying reagents
Summary
Membrane proteins that reside on the cell surface of leucocytes contain many cysteine (Cys) residues that mainly exist in an oxidized redox state as disulfide bonds. Structural disulfide bonds are usually buried inside the core of a protein or protein domain such as those found in the core of the immunoglobulin (Ig) fold. These structural disulfide bonds are protected from reduction by small molecule and enzymatic reducing agents that can be present in the extracellular space. It has become clear that there are disulfide bonds present in cellsurface proteins that are involved in regulating molecular function upon reduction to their constituent Cys residues. In order for a disulfide bond to be redox-labile, it has to be accessible to reducing
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